The use of microorganisms for the production of useful polypeptide products through recombinant DNA technology is becoming established as an industry. Foreign genetic material may be introduced into a culture of microorganisms, and, given the proper intracellular and extracellular conditions, the desired protein product(s) may be synthesized from the foreign gene(s). Such genetic material is commonly introduced into microorganisms in the form of plasmids, which are autonomously replicating extrachromosomal elements. In order to ensure the maintenance of plasmids within a culture of transformed cells, it has been necessary to grow those cells under special conditions. In the absence of such conditions, the plasmids, which may be inherently unstable, will not be maintained, and the cell population will revert to the untransformed state.
Increased plasmid stability and copy number are important to the biotechnology industry as a means of maintaining the production of plasmid-encoded proteins at a consistently high level. Previously reported attempts to increase plasmid stability do not appear to be optimal for commercial application. The introduction of yeast centromeres into ARS-bearing plasmids, while enhancing stability, has been shown to markedly decrease plasmid copy number (Clarke and Carbon, Nature 287:504-509, 1980 and Stinchcomb, et al., J. Molec. Biol. 158:157-179, 1982). Linear centromeric yeast plasmids similarly show an inverse relationship between stability and copy number (Murray and Szostak, Nature 305:189-193, 1983).
Plasmids typically contain gene sequences, known as selectable markers, which encode antibiotic resistance or complement nutritional requirements of the host cell. To select for the presence of such plasmids, transformed cells must thus be grown in special media which contain a selective drug or which are depleted for specific nutrients. These media requirements may be both expensive and prohibitive of optimal cell growth rates during the large-scale fermentation process. Many of such plasmids have been reported in the literature. Those comprising antibiotic drug resistance genes include pBR322 (Bolivar, et al., Gene 2:95-113, 1977) and its derivatives, such as the pUC vectors (Vieira and Messing, Gene 19:259-268, 1982) which carry a gene for ampicillin resistance; and pBR325 (Prentki, et al., Gene 14:289, 1981) which carries resistance genes for ampicillin, tetracycline, and chloramphenicol. Plasmids which complement host nutrient requirements include the yeast vectors YEp13 (Broach, et al., Gene 8:121-133, 1979), which carries the LEU2 gene; and YRp7' (Stinchcomb, et al., Nature 282:39, 1979), which carries the TRP1 gene. Mammalian cell selection systems include the use of the dihydrofolate reductase (DHFR) gene which confers resistance to methotrexate (reviewed by Schimke, Cell 37:705-713, 1984) and antibiotic resistance, such as resistance to G418 (Southern and Berg, J. Mol. Appl. Genet. 1:327-341, 1982).
It is therefore an object of the present invention to provide DNA constructs containing, as selectable markers, gene sequences whose products are essential for the viability or normal growth of the host cell on complex media.
It is another object of the present invention to provide recombinant host cells containing DNA constructs which are selectable by growth on complex media.
It is a further object of the present invention to provide cells deficient in essential functions which may act as hosts for DNA constructs carrying gene sequences which complement these defective essential functions.
It is yet another object of the present invention to provide methods for producing foreign proteins in recombinant host cells, wherein the proteins are the products of genes carried on DNA constructs which contain, as selectable markers, gene sequences which complement a deficiency in an essential gene in the host cells.
Other objects of the invention will become apparent to those skilled in the art.